Abstract
Streptococcus constellatus, a member of the Streptococcus anginosus group (SAG), is a Gram-positive bacterial species commonly found in the oral and gastrointestinal tracts; however, it can become pathogenic, particularly in immunocompromised individuals. S. constellatus is associated with the development of purulent infections that typically require a combination of antibiotic therapy and surgical intervention for effective treatment. This understudied emerging pathogen appears to possess a comABCDE quorum sensing (QS) circuitry, which is common in many streptococci species; however, the molecular interactions that drive this circuitry and its regulatory function remain unexplored. Herein, we confirmed the identity of the S. constellatus competence-stimulating peptide (CSP) and performed phenotypic assays to investigate its regulatory role in competence and biofilm development. To examine the role of each amino acid within the CSP sequence in QS activity, we conducted d-amino acid and alanine scans of the S. constellatus CSP. Given the uniquely high natural QS induction observed in S. constellatus, we developed a ΔcomC luciferase reporter system to assess the activity of the d-amino acid and alanine scan analogs on the competence regulon. Our results revealed that a single Alanine substitution could lead to a 60-fold increase in CSP potency. Furthermore, through circular dichroism (CD) analysis, we investigated the correlation between the secondary structure and biological activity. Overall, this study aims to enhance our understanding of S. constellatus, a relatively understudied species with pathogenic potential, and aid in the development of strategies to mitigate its pathogenicity.